Inactivation kinetics of food enzymes during ohmic heating

Alina Jakb, Jolanta Bryjak, Halina Wjtowicz, Viera Illeov, Jlius Annus, Milan Polakovic
*
Department of Chemical and Biochemical Engineering, Institute of Chemical and Environmental Engineering, Faculty of Chemical and Food Technology,
Slovak University of Technology in Bratislava, Radlinskho 9, 812 37 Bratislava, Slovak Republic
a r t i c l e i n f o
Article history:
Received 21 July 2009
Received in revised form 15 March 2010
Accepted 21 April 2010
Keywords:
Ohmic heating
Food pasteurisation
Alkaline phosphatase
Pectin methylesterase
Peroxidase
Inactivation mechanism
Mathematical modelling
a b s t r a c t
Ohmic heating of milk and fruit and vegetable juices was carried out at several incubation temperatures
to investigate inactivation of alkaline phosphatase, pectin methylesterase and peroxidase. Mechanisms of
inactivation of these enzymes and corresponding kinetic models were veried for each food material,
using the multitemperature evaluation of inactivation data. Compared to inactivation by conventional
indirect heating, kinetic parameters were changed but inactivation mechanisms remained the same.
The kinetic parameter changes were relatively minor for pectin methylesterase and alkaline phosphatase.
A signicant destabilization of the labile isozyme fraction of peroxidase occurred by the effect of ohmic
heating when the greatest decrease of stability was obtained for carrot juice.
2010 Elsevier Ltd. All rights reserved.
1. Introduction
Ohmic heating of food products involves the passage of alter-
nating electrical current through them, thus generating internal
heat as the result of electrical resistance (Reznick, 1996). The
amount of heat is directly related to the current induced by the
voltage gradient in the eld and the electrical conductivity of the
food material (Sastry & Li, 1996). This technology provides rapid
and uniform heating when the absence of a hot surface in ohmic
heating reduces fouling problems and thermal damage to a
product (Sastry & Barach, 2000). Food, which contains water and
ionic salts in abundance, is the most suitable for ohmic heating
(Palaniappan & Sastry, 1991).
Ohmic heating applications have a tremendous potential in
such elds of food processing technology as preheating, blanching,
sterilization, extraction, evaporation or dehydration. Ohmic
heating was used, for example, to increase the efciency of sucrose
extraction from sugar beet (Lima, Heskitt, & Sastry, 2001) or to
enhance the diffusion of soy milk from soybeans (Kim & Pyun,
1995). Its advantages, compared to conventional heating, include
maintaining the colour and nutritional value of food, shorter
processing times and higher yields (Castro, Teixeira, Salengke,
Sastry, & Vicente, 2004; Leizerson & Shimoni, 2005; Vikram,
Ramesh, & Prapulla, 2005; Wang & Sastry, 2002).
Since ohmic heating technology appears to be promising, its
effects on the quality parameters of food materials, such as inacti-
vation of microorganisms and enzymes or decomposition of heat-
sensitive compounds, should be investigated. There are very few
scientic reports dealing with these aspects of ohmic heating and
their comparison with the effects of conventional pasteurisation.
Castro, Teixeira, et al. (2004) studied the degradation of vitamin
C in strawberry products subjected to ohmic and conventional
heating. The obtained kinetic parameters were identical for both
heating processes which led to the conclusion that the presence
of electric eld did not affect the ascorbic acid degradation.
A continuous alternating current electric eld was applied to or-
ange juice containing Bacillus subtilis spores to examine its inacti-
vation. A pressurised electric sterilization system, using a
combination of high temperature and high electric eld, caused
effective inactivation of spores in a shorter time, with a smaller
loss of ascorbic acid and a less peculiar smell than with conven-
tional heating treatment (Uemura & Isobe, 2003).
Electrical elds, applied during ohmic heating of lipoxygenase
and polyphenoloxidase, caused their faster inactivation than dur-
ing conventional heating (Castro, Macedo, Teixeira, & Vicente,
2004). Similarly, ohmic heating was found to be more efcient
for the required microbial and pectin esterase inactivation due to
a shorter residence time when released avour compounds were
not degraded as quickly as during conventional pasteurisation
(Leizerson & Shimoni, 2005). Peroxidase in pea puree was also
inactivated in a shorter processing time, by ohmic heating, than
by conventional heating (Iier, Yildiz, & Baysal, 2006). Moreover,
ohmic heating caused less browning. The effects of voltage gradi-
ent, temperature and holding time on the polyphenoloxidase activ-
ity were investigated for grape juice ohmic heating (Iier, Yildiz, &
0308-8146/$ - see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2010.04.047
* Corresponding author. Tel.: +421 2 59325254; fax: +421 2 52496920.
E-mail address: milan.polakovic@stuba.sk (M. Polakovic).
Food Chemistry 123 (2010) 369376
Contents lists available at ScienceDirect
Food Chemistry
j our nal homepage: www. el sevi er . com/ l ocat e/ f oodchem
Baysal, 2008). The inactivation kinetics of this enzyme were de-
scribed by several mathematical models but simple rst-order
kinetics were found to be the best.
The objective of this study was to investigate the effect of ohmic
heating on the inactivation of three selected enzymes in food prod-
ucts and to compare it with the thermal inactivation of these en-
zymes under conventional indirect heating by hot water, which
was reported in our previous studies (Poata, Wilin ska, Bryjak, &
Polakovic, 2009; Wilin ska, Bryjak, Illeova, & Polakovic, 2007;
Wilin ska, de Figueiredo Rodrigues, Bryjak, & Polakovic, 2008).
Alkaline phosphatase (ALP; EC 3.1.3.1), peroxidase (POD; EC
1.11.1.7) and pectin methylesterase (PME; EC 3.1.1.11) are native
enzymes naturally present in milk, vegetables and fruits, respec-
tively. Their thermal resistance is greater than that of the most
heat-resistant non-spore-forming pathogens commonly found in
food (Grifths, 1986; Lemos, Oliveira, & Saraiva, 2000; Valdramidis
et al., 2007); therefore, they are used in standard tests for the
determination of proper food pasteurisation (Iier et al., 2006;
Painter & Bradley, 1997; Valdramidis et al., 2007). This study al-
lowed us to identify possible inactivation mechanisms of these en-
zymes and to evaluate the corresponding kinetics for ohmic and
conventional heating.
2. Materials and methods
2.1. Preparation of food materials
Fresh apple juice was obtained from Fructop, Ostratice (Slova-
kia). It was produced from three apple varieties: Golden Delicious
(65%), ampion (25%) and Rubinola (10%). The pH, saccharide con-
centration and total soluble solids were 3.55, 124 g/l and
13.46 Brix, respectively.
Two batches of raw cloudberry jam were obtained from Bltand
AB, Sweden, which differed in the contents of fruit components:
industrial standard (35% cloudberries, 58% sugar added and 7%
water added) and consumer type (70% cloudberries and 30% sugar
added). The jams were homogenised with a blender and obtained
fruit mash was centrifuged (10 000 rpm; 20 min; 4 C). A prepara-
tion of exogenous pectin methylesterase from Aspergillus niger
(Pectinase; Novozymes, Bagsvaerd, Denmark) was added in the ra-
tio 1:25 to the apple and cloudberry juices.
Bovine milks (raw and UHT-sterilized) were supplied by Rajo,
a.s. (Bratislava, Slovakia) and caprine milk was from Quinta dos
Moinhos Novos-Lacticnios, Lda. (Pvoa de Lanhoso, Portugal). In
order to obtain the initial activity values in the upper range of a
standard curve, the raw bovine milk was diluted with the UHT milk
in the ratio 1:40. Similarly, the raw caprine milk was diluted with
the caprine milk sterilized at 110 C for 20 min.
Three vegetables mixtures, based on carrot, broccoli and pota-
toes, were prepared according to the recipes provided by Natures
Best (Drogheda, Ireland). The main vegetable components (97.4%)
were mixed with butter (0.37%), milk (2.1%), salt (0.13%) and pep-
per (0.03%). All ingredients were mixed, chopped with a blender
and squeezed in a juice extractor.
All food materials were stored in a freezer at 22 C if they were
not used immediately after preparation; they were thawed before
use and were left to stand for one hour to release trapped air.
2.2. Assays of enzyme activity
2.2.1. Pectin methylesterase (PME)
The determination of the residual PME activity after thermal
treatment was based on the NaOH titration of formed carboxylic
groups of apple pectin substrate, which is described in detail
in our previous publications (Jakb, Bryjak, & Polakovic, 2009;
Wilin ska et al., 2008). The assay was carried out at 30 C and pH
4.5, using an automatic pH-stat.
2.2.2. Alkaline phosphatase (ALP)
The ALP activity was determined using a standard method
(Wilin ska et al., 2007); phenol was liberated from a disodium
phenyl phosphate substrate under controlled pH and temperature
and reacted with 2,6-dichloroquinone-4-chloroimide to form indo-
phenol blue, which was measured colorimetrically at 620 nm.
2.2.3. Peroxidase (POD)
A continuous spectrophotometric method was applied to the
determination of POD activity in vegetable juice supernatants that
was based on the recording of the increase of absorbance at
405 nm in an assay mixture containing a juice sample, solutions
of hydrogen peroxide and ABTS [2,2
0
-azinobis-(3-ethyl benzthiazo-
line-6-sulphonic acid)] as substrates (Poata et al., 2009).
2.3. Ohmic heating inactivation experiments
All ohmic heating experiments were conducted in a horizontal
cylindrical glass reactor of own construction with the inner diam-
eter of 2.4 cm and a length of 6.0 cm. The working volume, lled
with a heated material, was capped by stainless steel electrodes.
The temperature was measured online in the geometrical centre
of the reactor, using an axially placed platinum resistance ther-
mometer (Pt100). It was checked that the temperature gradients
inside the heated material were insignicant similarly, as in the
work of Zareifard, Ramaswamy, Trigui, and Marcotte (2003). The
heated material was stirred with a magnetic stirrer placed inside
the reactor at a frequency of 280 min
1
for PME and 135 min
1
for ALP and POD.
The ohmic heating system utilised an alternating current with a
frequency of 50 Hz and voltage adjustable by a power relay. The
intensity of heating was varied, based on measurements of current,
voltage and temperature. A system controller automatically deter-
mined the necessary voltage to reach a required temperature. Since
an objective was to compare the inactivation effects of conven-
tional and ohmic heating, the temperature during the initial heat-
ing period (Fig. 1) was controlled so that it simulated the
temperature course for conventional heating (Poata et al., 2009;
Wilin ska et al., 2007, 2008). The holding-phase temperature was
then controlled with an accuracy of 0.3 C.
Fig. 1. Thermal history of carrot mixture for conventional (h) and ohmic heating
(j).
370 A. Jakb et al. / Food Chemistry 123 (2010) 369376
Depending on the temperature used, thermal treatment was ap-
plied from 4 to 750 min in the case of ALP in milk, from 2 to
120 min for PME in fruit juices, and from 6 to 1500 min for POD
in vegetable juices. At different time intervals, 1 ml of sample
was taken through a sampling port in the reactor centre. The sam-
ple was immediately cooled for 5 min in an ice-water/ethanol mix-
ture (4 C) and then kept in an ice-water bath prior to activity
measurement.
2.4. Modelling
2.4.1. General
All models used in this work were applied in our previous stud-
ies for enzyme inactivation under conventional heating (Poata
et al., 2009; Wilin ska et al., 2007, 2008). Temperature courses were
calculated from the dynamic enthalpy balance:
dT
dt
KT
H
T 1a
t 0; T 298:15 K 1b
where T is the sample temperature, T
H
the set holding-phase tem-
perature, t the heating time and K is the proportionality factor,
which was taken for each material from the conventional heating
experiments.
In each model, the temperature dependence of the rate con-
stants of individual reactions was expressed by the Arrhenius
equation:
k
i
k
i0
e
E
ai
RT0
1
T
0
T

; i 14 2
where k
i
represents a rate constant, k
i0
its value at the reference
temperature of T
0
, E
ai
the activation energy, and R is the universal
gas constant.
The experimental data of pectin methylesterase, alkaline phos-
phatase and peroxidase inactivation, obtained at different temper-
atures, were modelled simultaneously using the so-called
multitemperature evaluation (Vrbel, Polakovic, tefuca, & Ble,
1997). The parameters of the individual models, the rate constants
at the reference temperature and activation energies were esti-
mated using the software Athena Visual Workbench (Stewart and
Associates Engineering Software, Madison, WI, USA).
2.4.2. Pectin methylesterase
PME inactivation kinetics were well described by the rst-order
model (Wilin ska et al., 2008), which was based on the one-step
irreversible reaction mechanism:
E!
k
1
I 3a
where E is the native form of the enzyme and I its inactive form. The
corresponding model equation was:
dC
E
dt
k
1
C
E
3b
where C
E
is the concentration of the native form. The differential
equation was solved with the initial condition:
t 0; C
E
C
E0
3c
The initial active form concentration C
E0
was also used to dene the
relative enzyme activity, a:
a
C
E
C
E0
e
k
1
t
3d
2.4.3. Alkaline phosphatase
The simple exponential model (Eq. (3d)) was used for caprine
milk alkaline phosphatase (Wilin ska et al., 2007) but, for bovine
milk alkaline phosphatase, a representative model was formulated
on the basis of the two-step LumryEyring mechanism (Lumry &
Eyring, 1954) where an intermediate denatured form, D, is revers-
ibly formed:
0 20 40 60 80 100 120
0.0
0.2
0.4
0.6
0.8
1.0
0 5 10 15 20
0.0
0.2
0.4
0.6
0.8
1.0
R
e
l
a
t
i
v
e

a
c
t
i
v
i
t
y

o
f

P
M
E
Time [min]
R
e
l
a
t
i
v
e

a
c
t
i
v
i
t
y

o
f

P
M
E
Time [min]
a
b
c
Fig. 2. Thermal inactivation of PME in cloudberry juice prepared from (a) industrial
jam and (b) consumer jam and (c) in apple juice at different temperatures: (j)
54 C, (d) 56 C, (h) 60 C, (N) 64 C and (s) 66 C. The solid lines represent the ts
of the data with the rst-order model obtained by multitemperature evaluation.
The inset plots present the inactivation courses at shorter times.
A. Jakb et al. / Food Chemistry 123 (2010) 369376 371
E
k
1
k
2
D!
k
3
I 4a
The corresponding model equations were as follows:
dC
E
dt
k
1
C
E
k
2
C
D
4b
dC
D
dt
k
1
C
E
k
2
k
3
C
D
4c
t 0; C
E
C
E0
C
D
0 4d
2.4.4. Peroxidase
The inactivation process of peroxidase under conventional heat-
ing appeared to have a biphasic character in each vegetable juice
(Poata et al., 2009). For broccoli peroxidase, this could be de-
scribed with a very good accuracy by the simple isozyme mecha-
nism according to the scheme:
E
1
!
k
1
I
1
5a
E
2
!
k
2
I
2
5b
where E
1
and E
2
are native isoforms, and I
1
and I
2
their inactive
forms. The mathematical model consisted of ordinary differential
equations describing the changes of C
E
1
and C
E
2
:
dC
E
1
dt
k
1
C
E
1
5c
dC
E
2
dt
k
2
C
E
2
5d
To describe carrot and potato inactivation data, the above iso-
zyme mechanism was extended so the simple irreversible one-step
reaction of one isoform was replaced by the series mechanism of
LumryEyring:
E
1

k
1
k
2
D!
k
3
I
1
6a
E
2
!
k
4
I
2
6b
The model equations describing the concentration changes of spe-
cies were as follows:
dC
E
1
dt
k
1
C
E
1
k
2
C
D
6c
dC
D
dt
k
1
C
E
1
k
2
C
D
k
3
C
D
6d
dC
E
2
dt
k
4
C
E
2
6e
The concentrations in Eqs. (5c), (5d) and (6c)(6e) were substi-
tuted by relative activities, which were obtained after the multipli-
cation of the equations by the corresponding molar activities of the
enzymatic forms and the division by the total initial activity. An
exception is the inactive form D, whose activity was formally ob-
tained as a product of its concentration and molar activity of E
1
.
The initial conditions for both sets of differential equations were:
t 0; a
E
1
a; a
E
2
1 a; a
D
0 7
where a
E
1
, a
E
2
and a
D
are the relative activities of the forms E
1
, E
2
and D, respectively. The fraction of the initial relative activity of iso-
form E
1
, a, was a tted model parameter.
3. Results and discussion
3.1. Pectin methylesterase
Pectin methylesterase (EC 3.1.1.11) is an enzyme that has been
found in essentially every plant tissue, several fungi and bacteria.
PME has no prostetic group and catalyses deesterication of
galactosyluronate methylesters of pectins, releasing protons and
methanol into the media. Fruit PME activity is responsible for
clarication of juices and wines and for reduction of viscosity.
For that reason, PME is commonly used for improving the texture
and rmness of processed fruit, as well as in the extraction and
clarication of fruit juices. Commercial preparations of PME are
produced from fungal sources, especially from A. niger (Jayani,
Saxena, & Gupta, 2005).
The inactivation of exogenous pectin methylesterase in apple
juice and cloudberry jams has been found to follow rst-order
kinetics (Wilin ska et al., 2008). The same trend was observed in
this work when experiments for the same materials were carried
out under ohmic heating (Fig. 2ac). The same intervals of temper-
ature were applied as for conventional heating experiments, which
implies that the rate of inactivation was not very different. The
inactivation curves were evaluated by the multitemperature meth-
od, using the rst-order kinetic model described by Eqs. (1)(3).
Fig. 2ac show that a good agreement of experimental and model
Table 1
Comparison of kinetic parameters of PME inactivation in different food materials for conventional and ohmic heating.
Parameter Cloudberry jam industrial standard Cloudberry jam consumer type Apple juice
Conventional heating Ohmic heating Conventional heating Ohmic heating Conventional heating Ohmic heating
k
10
(min
1
) 0.257 0.010 0.330 0.014 0.327 0.009 0.428 0.019 0.093 0.003 0.136 0.006
E
a1
(kJ mol
1
) 284 7.10 275 8.00 281 5.87 283 8.52 309 6.33 328 8.80
s
a
0.035 0.034 0.027 0.038 0.035 0.043
The values after the plus/minus sign represent the half-widths of 95% condence intervals.
s
a
mean error of relative activity.
T
0
= 333.15 K.
326 328 330 332 334 336 338 340
1
0.1
0.01
Fig. 3. Comparison of temperature dependence of rate constants of PME inactiva-
tion in a juice from consumer-type cloudberry jam under conventional (solid line)
and ohmic heating (dotted line) (with reciprocal horizontal and logarithmic vertical
axes).
372 A. Jakb et al. / Food Chemistry 123 (2010) 369376
activity values was achieved. This agreement is also conrmed by
the mean errors of activity presented in Table 1. They were
approximately the same as those previously reported in the work
of Wilin ska et al. (2008).The estimated model parameters are
presented in Table 1 where the values for conventional heating
inactivation are included for comparison. Fig. 3 compares the dif-
ference between the effects of conventional and ohmic heating
on the temperature dependences of inactivation rate constants
for consumer-type cloudberry jam. Table 1 shows that the values
of the activation energy for different materials were all about
300 kJ mol
1
. This value means that the rate constants were dou-
bled with an increase of temperature of about 2 C (Fig. 3). The dif-
ferences between the activation energy values for ohmic and
conventional heating were within the error of estimation. Table 1
and Fig. 3 reveal that pre-exponential factors in the Arrhenius
equation were always higher for ohmic heating; the factor was
about 3040%. This difference corresponds to a temperature incre-
ment DT lnk
i0;ohm
=k
i0;conv
RT
2
=E
ai
of about 0.8 C. If the accu-
racy of temperature measurements and control in two different
heating systems is considered, it can be concluded that almost
the full extent of activity loss with ohmic heating was caused by
the Joule heat effect. Only a very small contribution of effects such
as modication of the enzyme surface charge or enzyme environ-
Table 2
Comparison of kinetic parameters of milk ALP inactivation for conventional and
ohmic heating.
Parameter Bovine milk Caprine milk
Conventional
heating
Ohmic
heating
Conventional
heating
Ohmic heating
k
10
(min
1
) 0.089 0.005 0.156 0.014 0.112 0.006 0.163 0.004
k
20
(min
1
) 0.029 0.014 0.143 0.081
k
30
(min
1
) 0.072 0.031 0.237 0.105
E
a1
(kJ mol
1
) 376 8.12 331 11.5 379 8.8 447 6.4
E
a2
(kJ mol
1
) 303 78.8 506 126
E
a3
(kJ mol
1
) 416 74.9 749 135
s
a
0.017 0.017 0.036 0.022
The values after the plus/minus sign represent the half-widths of 95% condence
intervals.
s
a
mean error of relative activity.
T
0
= 331.15 K.
a
b
Fig. 4. Thermal inactivation of (a) bovine and (b) caprine milk ALP at different temperatures: (j) 52 C, (d) 55 C, (h) 58 C, (N) 61 C and (s) 64 C. The solid lines represent
the ts of the data with the LumryEyring (a) and rst-order models (b) obtained by multitemperature evaluation. The inset plots present the inactivation courses at shorter
times.
A. Jakb et al. / Food Chemistry 123 (2010) 369376 373
ment by ionisation of solution components could be inferred from
these results.
3.2. Alkaline phosphatase
Alkaline phosphatase (EC 3.1.3.1) is a membrane-bound glyco-
protein, widely distributed in animal tissues and microorganisms.
Milk ALP is a dimeric metalloenzyme comprised of two similar
subunits of molecular weight of about 85 kDa. The enzyme re-
quires two metals for maximal activity: zinc is essential and mag-
nesium is stimulatory (Castro, Swanson, Barbosa-Cnovas, &
Meyer, 2001).
A study of the kinetics of thermal inactivation of milk ALP under
conventional heating showed that the inactivation of caprine milk
ALP followed the rst-order kinetics model, like PME in fruit juices,
whereas bovine milk ALP inactivation was described by a two-step
model, based on the LumryEyring mechanism (Wilin ska et al.,
2007).
The milk ALP activity loss under ohmic heating was measured
in the temperature range of 5264 C (Fig. 4a and b). The inactiva-
tion experiments were modelled using Eqs. (1), (2) and (4) for bo-
vine milk ALP and (1)(3) for caprine milk ALP. Good ts of the
experimental data with these models are illustrated in Figs. 6
and 7. Table 2 shows that the activity mean error was equal or low-
er as that for conventional heating inactivation where a complete
statistical analysis of parameter estimation was made (Wilin ska
et al., 2007).
Table 2 also presents the estimated parameters for both en-
zymes. In the case of caprine milk ALP, the values of both the rate
constant k
10
and activation energy E
a1
were higher for ohmic heat-
ing than for conventional heating. The former parameter was high-
er by 45% and the latter one by 18%. The Arrhenius plots of the rate
constant k
1
displayed in Fig. 5, however, show that the rate of inac-
tivation was approximately the same for these two heating modes
at the lowest temperature. The higher activation energy estimated
for ohmic heating resulted in the increase of the difference be-
tween the inactivation rate constants for ohmic and conventional
heating with temperature. But, even the difference of reaction rates
at the highest temperature was not very large; it corresponded to a
temperature increment of 0.8 C. The principal conclusion is then
the same as for PME inactivation, that almost the full extent of
activity loss under ohmic heating was caused by the Joule heat
effect.
It is much more difcult to compare the parameters of the
LumryEyring model for bovine milk ALP inactivation presented
in Table 2. It is evident that all parameter values are higher for
ohmic heating. It should, however, be emphasised that the increase
0 100 200 300 400
0.0
0.2
0.4
0.6
0.8
1.0
0 5 10 15 20 25 30 35 40
0.0
0.2
0.4
0.6
0.8
1.0
R
e
l
a
t
i
v
e

a
c
t
i
v
i
t
y

o
f

P
O
D
Time [min]
R
e
l
a
t
i
v
e

a
c
t
i
v
i
t
y

o
f

P
O
D
Time [min]
a
b
c
Fig. 6. Thermal inactivation of (a) broccoli, (b) potato and (c) carrot POD at different
temperatures: (j) 58 C, (d) 62 C, (h) 66 C, (N) 70 C, (s) 74 C and (4) 78 C. The
solid lines represent the ts of the data with the simple (a) and extended (b) and (c)
isozyme models obtained by multitemperature evaluation. The inset plots present
the inactivation courses at shorter times.
Fig. 5. Comparison of temperature dependence of rate constants of caprine milk
ALP inactivation under conventional (solid line) and ohmic (dotted line) heating
(with reciprocal horizontal and logarithmic vertical axes).
374 A. Jakb et al. / Food Chemistry 123 (2010) 369376
of the rate constant, k
20
, of the backward reaction, represents a
shift of the equilibrium of the reversible reaction toward the native
enzyme. Moreover, as was pointed out in our previous paper
(Wilin ska et al., 2007), the uncertainties and cross-correlation of
parameters are signicant, partly caused by a relatively small
deviation from rst-order kinetics. The lowest uncertainties were
associated with the parameters k
10
and activation energy E
a1
. If
these are compared for ohmic and conventional heating and the
reversible character of the reaction is considered, an identical con-
clusion for the effect of ohmic heating is obtained as for caprine
ALP.
3.3. Peroxidase
Peroxidase (EC 1.11.1.7) is a heme-containing enzyme which
can catalyse a large number of reactions in which a peroxide is re-
duced while an electron donor is oxidised. Peroxidase activity in
raw and unblanched vegetables is responsible for off-avours
and off-colours. PODs are present in plant tissues in the form of
several isoenzymes varying in substrate specicity, thermal stabil-
ity, molecular weight, isoelectric point and immunological proper-
ties. The thermal stability of vegetable PODs varies considerably
with the source and origin (Robinson, 1991).
Our previous study dealt with thermal inactivation kinetics of
broccoli, carrot and potato POD under conventional heating (Poata
et al., 2009). Broccoli POD inactivation kinetics were described by a
simple isozyme mechanism whereas, in the case of carrot and
potato POD, the inactivation of one isozyme was described by
the LumryEyring mechanism. In this work, ve ohmic heating
inactivation experiments were carried out for the same vegetable
mixtures (Fig. 6ac). The temperature range applied was 58
74 C for broccoli and potato mixtures and 6278 C for a carrot
mixture since its POD was more thermally stable.
A visual comparison of the inactivation curves revealed that
POD inactivation proceeded clearly faster under ohmic heating
than under conventional heating in the second phase whereas, in
the rst phase, minimal differences were observed. This is well
illustrated in Fig. 7 for carrot POD inactivation at 70 C.
The inactivation experiments presented in Fig. 6ac were mod-
elled with the same models as those applied for conventional heat-
ing inactivation experiments (Poata et al., 2009). The model for
broccoli POD inactivation was formed from Eqs. (1), (2), (5) and
(7) and the model for potato and carrot POD inactivation was
formed by Eqs. (1), (2), (6) and (7). A good quality of the ts with
the models is illustrated in Table 3 when the activity mean errors
were even slightly lower than those for conventional heating inac-
tivation, for which a complete statistical analysis of parameter esti-
mation was made (Poata et al., 2009).
The analysis of parameter values of the models of POD inactiva-
tion presented in Table 3 faces the same problem as described
above for the model of bovine ALP inactivation. The uncertainties
and cross-correlation of parameters are rather large for most of
the parameters. One exception is the parameter a. Its values for
all PODs were the same as for conventional heating if the error
of estimation is considered. The initial fraction of labile isozyme
was around 30% for all vegetable materials. As far as the rate con-
stants are concerned, the most reliable estimates were obtained for
the parameters related to the irreversible reaction of the stable iso-
zyme, which are k
20
and E
a2
for broccoli POD and k
30
and E
a3
for po-
tato and carrot PODs.
Table 3 shows that the activation energies of the irreversible
reaction were essentially the same for ohmic and conventional
heatings but the rate constants at the reference temperature were
different. This would mean that Arrhenius plots for ohmic and con-
ventional heating would be parallel lines, as was illustrated for
PME in Fig. 3. The distances between these lines would be larger
than that for PME because, under ohmic heating, the rate constant,
k
30
, for potato POD was twice larger, k
20
for broccoli POD was three
times larger, and k
30
for carrot POD was 10 times larger. The corre-
sponding temperature shifts in thermal stability of the enzyme
were 2 C for potato POD, 3.4 C for broccoli POD and 8 C for carrot
POD.
In contrast to PME and ALP, ohmic heating caused a signicant
entropic effect on the rate of inactivation of POD. Since the activa-
tion enthalpy, which in the Eyring theory is the equivalent of
activation energy, was the same for ohmic and conventional hea-
Fig. 7. Carrot peroxidase inactivation under conventional (h) and ohmic (j)
heating at 70 C.
Table 3
Comparison of kinetics parameters of POD inactivation for conventional and ohmic heating.
Parameter Broccoli Potato Carrot
Conventional heating Ohmic heating Conventional heating Ohmic heating Conventional heating Ohmic heating
k
10
(min
1
) 0.264 0.057 0.153 0.055 42.5 25.2 22.7 20.0 3.48 1.81 3.89 5.37
k
20
(min
1
) 0.015 0.001 0.044 0.005 12.7 8.08 12.4 12.4 3.78 2.17 6.81 10.7
k
30
(min
1
) 0.006 0.002 0.012 0.003 0.005 0.001 0.051 0.012
k
40
(min
1
) 8.29 3.74 10.9 4.49 0.254 0.044 0.499 0.177
a 0.264 0.022 0.257 0.105 0.689 0.035 0.599 0.064 0.692 0.025 0.698 0.060
E
a1
(kJ mol
1
) 70.7 34.2 198 47.2 182 30.1 173 44.2 100 31.5 162 89.9
E
a2
(kJ mol
1
) 332 6.1 302 13.4 192 33.5 169 47.8 104 33.0 165 83.6
E
a3
(kJ mol
1
) 301 48.3 339 41.3 358 20.8 271 41.1
E
a4
(kJ mol
1
) 379 68.2 343 69.8 330 35.1 128 78.8
T
0
(K) 339 339 339 339 343 343
s
a
0.023 0.029 0.022 0.027 0.017 0.021
The values after the plus/minus sign represent the half-widths of 95% condence intervals.
s
a
mean error of relative activity.
A. Jakb et al. / Food Chemistry 123 (2010) 369376 375
tings, no direct effect of ohmic heating on the enzyme conforma-
tion can be inferred. The destabilization effect of ohmic heating
may thus be associated with the modication of enzyme surface
charge and/or enzyme environment by ionisation of solution com-
ponents and distribution of their ions in an electric eld. The re-
sults also imply that the destabilizing effect may be stronger for
a denaturation intermediate than for the native stable isozyme.
4. Conclusion
This study shows that ohmic heating, which can be a useful
alternative method for sterilization or pasteurisation of food prod-
ucts, may also enhance the rate of inactivation of enzymes present
in food materials. Some, albeit slight, decrease of enzyme stability
may occur for any food material, as was shown here for pectin
methylesterase in fruit juices and alkaline phosphatase in milk.
By contrast, peroxidase in vegetable juices is more prone to desta-
bilization by ohmic heating.
A thorough kinetic study of enzyme inactivation is a very useful
tool to analyse the effects of ohmic heating when a Joule heat con-
tribution should be distinguished from other factors affecting
activity loss. The method of multitemperature evaluation resulted
in a conclusion that the mechanism of any enzyme studied is not
changed compared to conventional heating. The same form of ki-
netic equations can be applied but their parameters are changed.
Another important observation made is that only the activation en-
tropy, but not the activation enthalpy, is different for ohmic heat-
ing. This implies that a cause of the decreased stability does not lie
in the modication of enzyme tertiary structure by the electric
eld. It is assumed that it can be explained by the affect of modied
environment due to the increased number of ions and their differ-
ent distributions around enzyme molecules.
Acknowledgement
This study was supported by grants from the 6th Framework
Programme of EU, Project FOODPRO (Ohmic heating for food pro-
cessing), No. SME-2003-1-508374.
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